CANCER BIOMARKER DETECTION STRATEGIES TO DIRECT PRECISION 

MEDICINE

CANCER BIOMARKER DETECTION STRATEGIES TO DIRECT PRECISION 

MEDICINE

GLORIA RIBAS, PhDDays on Personalised Medecine, Wien, Austria

24‐25 February, 2017

PERSONALIZED MEDICINE

“One size fits all” treatmentshave become obsolete

SCHEDULING OF TUMOR BIOPSIES AND THE OPORTUNITIS FOR GENOMIC ANALYSIS

Dienstmann R, Rodón J, Tabernero J. J Clin Oncol 2013 Von Hoff D, et al. J Clin Oncol 2010 

SIGNALLING PATHWAYS ALTERED IN CANCER

MUTATIONS IN ONGOGENE PI3KCA

RESISTANCE TO HER2 BREAST CANCER TREATMENT

IN SEARCH OF NEW THERAPIES

LESS COMPLETE PATHOLOGICAL RESPONSES 

GOOD RESPONSES

BREAST CANCERER/PGR +HER2+

PIK3CAMUTATIONS

NEO‐ADYUVANCY

COLORECTAL CANCER

anti‐EGFR therapiesCetuximab, panitumumab

NON respondants

MUTATIONSKRAS

40‐50%

+ QMT

Metastasic Cancer CRC

respondants

IMPROVED SURVIVAL

We are participating in a H2020‐Motricolor proyect with three clinical trials based onmolecular profiling. Patients will have dedicatedtherapies according to their genomic profile.

LUNG CANCER

NSCLC 80%

anti‐EGFR THERAPIESerlotinib o gefitinib

respondants

NO respondedores

Asian ethnicity,  female sex, adenocarcinoma histology,  light/never smokersAsian ethnicity,  female sex, adenocarcinoma histology,  light/never smokers

MUTATIONS (L858R)EGFR

AmplificationMET  (20%)

MUTATION T790M EGFR (50%)

MUTATIONK‐RAS /N‐RAS (20‐25%)/(1%)

TRASLOCATIONEML4‐ALK(1‐7%)

ALK‐TK inh THERAPIEScrizotinib

respondedores

EGFR mutations and K‐rasmutations are almost always mutually exclusive. 

the lack of response to an EGFR TKI is likely more closely linked and better predicted by the lack of an EGFR mutation than the existence of a K‐rasmutation.

ios ensayos clínicos novedosos

B-RAF

MUTATION V600 (*)B‐RAF

vemurafenib y  dabrafenib

respondants

LATE‐STAGE MELANOMA

Non respondants

Aprox 50% melanomas

MEK inhibitors, trametinib(Mekinist) y cobimetinib(Cotellic), show reduction of somemelanomas with BRAF mutations

MELANOMA

SETUP OF A PRESCREENING PROGRAM IN CANCER:

SAMPLESAMPLE

PLATFORM SELECTIONPLATFORM SELECTION

SCOPESCOPE

ROBUSTNESSROBUSTNESS

SENSITIVITYSENSITIVITY

TIMELINESTIMELINES

COSTCOST

SUCCES IN MATCHING PATIENTS TO THERAPYSUCCES IN MATCHING PATIENTS TO THERAPY

SOURCESOURCE

STORAGESTORAGE

TYPES OF BIOLOGICAL TISSUES

FFPE TMA

LIQUID BIOPSIES

FRESH FROZEN

‐BIOLOGICAL MATERIAL IN GOOD SHAPE FOR –ÓMIC STUDIESBEST QUALITY AND QUANTITY

‐ NOT ALWAYS STORED APPROPRIATELY, ‐NOT ALWAYS ACCESIBLE‐SINGLE POINT IN TIME

‐ MOST COMMONLY CONSERVED IN CLINICAL SETTINGS, (ALLOWS RETROSPECTIVE AND ARCHIVED MATERIAL).

‐ CONTAMINATED WITH NORMAL CELLS (MIN % SHOULD BE ESTABLISHED)

‐ EASY TO OBTAIN IN RUTINE CLNICAL ANALYSIS (WILL FACILITATE MONITORING DISEASE, 

‐ MINIMUN INVASIVE‐ CONTAINS (PLATELET DERIVED 

CELLS, INMUNE CELLS, AND IN THE SERUM (EXOSOMES, ctDNA, CTCs).

‐ SUBOPTIMAL EXTRACTION OF BIOLOGICAL MATERIAL (RNA, DNA, PROTEINAS) DEGRADED

‐ STATIC SAMPLE 

‐ LOW ABUNDANCE AND FRAGMENTED BIOLOGICAL MATERIAL BUT PRESENT

‐ REQUIRED ULTRASENSITIVE DETECTION METHODS

DISADVANTAGESADVANTAGES

SAMPLESAMPLE

CLONAL HETEROGENEITY

‐ TUMORS ARE CLONALLY HETEROGENEOUS‐ DIFFERENT SECTIONS CAN HAVE DIFFERENTS ALTERATIONS‐METASTASIS CAN DIFFER FROM PRIMARY TUMOR‐ TUMOR CAN EVOLVE AT PROGRESSION

Allison K., et al., Oncology Journal 2014Gerlinger, N Engl J Med 2012

SAMPLESAMPLE

TIMELINES

In a clinical scenario, timelines are very short. In Phase I trials, 2-3 weeks could be considered acceptable as total tournaround time

In a clinical scenario, timelines are very short. In Phase I trials, 2-3 weeks could be considered acceptable as total tournaround time

Sanger sequencing (several genes) can take up to 2 weeks

Assays developed to test 1 mutation in 1 oncogene can be done in < 1 week

Multiplex Assays can be done whithin 1 week

NGS for large list of candidate genes, of sequencing exomes o whole genomemay take 6-8 weeks. The bottle neck is in the analysis. Should not last for more than two weeks.

ROUTINE SCREENING PROGRAM AT OUR HOSPITAL

CANCER FX GENOMICS LABExtracts DNA

Quantifies and QCsPerforms mutation detection

Min 3-4 days(max 1 week)

1 weekx 8 patients

Total prescreening ProgramTurnaround time 2 weeks

CLINICIAN requestsFor somatic mutation profiling

MOLECULAR PATHOLOGY LABselects FFPE blocks

Evaluates tumor content and cuts slices

Results are manually curated

And are sent back to the clinician

PLATFORM SELECTION

SANGER SEQUENCING NGS JUNIOR ROCHE MASSARRAY‐SEQUENOM

NGS MISEQ, ILLUMINA DIGITAL PCR, SYSMEX‐INOSTICS

<2010 . . . . . . 2013 . . . . . . 2014 . . .

. . . 2016 . . . . . . 2017 . . .

SANGER SEQUENCING

- Quality- long reads- Costs- Analog trace peaks MIN ALLELE

Detection 25%

gDNA

Gene A Gene B

Long Fragments 150-800bp

One trace, from all fragments labelled

TYPE OF FUNCIONAL GENES – IMPORTANCE TO DESIGN ASSAYS

ONCOGENES: Hostspots

AKT1, BRAF, KRAS, PIK3CA, NRAS, HRAS . . .

ONCOGENES: Hostspots

AKT1, BRAF, KRAS, PIK3CA, NRAS, HRAS . . .

ONCOGENES: Hostspots

AKT1, BRAF, KRAS, PIK3CA, NRAS, HRAS . . .

SUPPRESORES DE TUMORES: great diversityof alterations

TP53, PTEN, P53, NF1, VHL . . .

indels

MUT

NGS FIRST GENERATION

Nº GENE CHR

1AKT1 14

2 BRAF 7

3 KRAS 12

4 KRAS 13

5 PIK3CA 3

6 PIK3CA 3

7 PIK3CA 3

8NRAS 1

9 EGFR 7

10 EGFR 7

11 EGFR 7

EXON LONG MUT

E3 125 p.E17K

E15 119 p.V600F

E2 122 p.G12+/p.G13+

E3 179 p.Q61+

E2* 428 p.E110K

E10* 125 p.e542+/p.E545+

E21* 6000 p.T1025+/p.H1047+

E2 128 p.G12+/p.G13+

E19 99 p.E747del

E20 186 p.T790M

E21 156 p.L858R

u

NEXT GENERATION SEQUENCING: NGS JUNIOR (ROCHE)

GENOTING Y GENETIC DIAGNOSTIC (UCIM, UV‐INCLIVA)6 GENES (AKT1, BRAF, KRAS, PIK3CA, NRAS and EGFR)

15 HOTSPOTS

MUTATION PRE‐SCREENING BY SEQUENOM

Fleitas‐et al. Cancer Treat Reviews, 2016

OncoCarta Panelv1:19 Oncogenes/238 hostspots

Clia‐Vall D’Hebron Panel: 5 additional oncogenes, 8 genes /86 hotspots

Incliva Panel: ERBB2,ERBB3,ERBB4  

TYPES OF TUMORS ANALYZED BY MASSARRAY TECHNOLOGY

Widely used technology

Mass spectrometry technology remains a good validation technologyand is optimal when only hotspots are pursued

Fleitas‐et al. Cancer Treat Reviews, 2016

MUTATED GENES AND SPECIFIC MUTATIONS FOUND

KRAS PIK3CA KIT RET BRAF EGFR MET NRAS

PIK3CA KRAS ABL1 AKT1 CDK1

EGFR GNAS KIT NRAS PDGFRA

35,5%

22,6%

35%

17%

A146VG12DG12SQ61K

TUMORES GASTROINTESTINAL TUMORSTUMORES GASTROINTESTINAL TUMORS

TUMORES BREAST/UTERUS/ENDOMETRYTUMORES BREAST/UTERUS/ENDOMETRY

G12SQ61R

G1049RC420RE542K

C634WC634Y

L597SV600E

D52N

R970CD770_N771>AGG

P772_H773insV

C420RE542KH1047R

G12D, G12DQ61K

G13DD1071N

E839K

R201H

R24C

E17K

Y253H

P772_H773insV

MUTATIONS FOUND ACROSS TUMOR SAMPLES 

197 samples analysed (49% HAD 1 MUTATION IN 16 DIFFERENT GENES% CRC, % Breast)‐Mutations in KRAS and PIK3CA were detected in 40/97 (41.2%)and 30/97 (30.9%) patients respectively. ‐Thirty‐one patients (32.0%) had mutations in two genes, ‐20 of them (64.5%) initially diagnosed with colorectal cancer ‐ co‐occurrence of mutation involved mainly KRAS, PIK3CA, ‐ KIT and RET

Ibarrola‐Villava et al., Oncotarget 2015

OPTIONS OF TARGETED THERAPIES

101 PATIENTS WERE CANDIDATES TO BENEFIT FROM TARGETED THERAPIES75 had actionable mutations26 were KRASwt and could have possible be treated with anti‐EGFR agents

5 anti‐EGFR15 OTHER THERAPIES8 were selected for clinical trials

5 got PI3K/AKT inhibitors1 anti‐IGF1, 2 anti ERBB3

34% HAD STANDARD THERAPY

26% DIS NOT PROGRESSWITH CURRENT THERAPY

28% ACTUALLY BENEFITED FROM TARGETED THERAPIES

(CO‐MORBIDITIES, POOR PERFORMANCE STATUS, CONCURRENT 2º `NEOPLASM,OR LOSS OF FOLLOW UP

TARGETED THERAPIES

Ibarrola‐Villava et al., Oncotarget 2015

THEY ARE MORE GENES MUTATED OUT THERE?

DISCOVERY AND SATURATION ANALYSIS OF CANCER GENES ACROSS 21 TUMOUR TYPESMS Lawrence et al. Nature 000, 1-7 (2014)

Cancer genes identified from a data set of 4,742 tumours. Down-sampling analysis shows that gene discovery is continuing as samples and tumour types are added.

GENOMIC PLATFORMS TO SCREEN A PANEL OF CANDIDATE GENES

masa

non extendedprimer

complete genome

Complete exome

“customized panels” 

MALDI‐TOFF

NGS

Method to selectwanted sections

Resequencing – candidate gene-exons approach

IILLUMINA SEQUENCING: MISEQ APPLICATIONS IN CANCER GENOMICS

NimbleGen Seq Capture

Hybridization

Bead capture

HaloPlex (Agilent)

EGFR L747P MUTATION

MUTATION DETECTION

DISCOVERY AND SATURATION ANALYSIS OF CANCER GENES ACROSS 21 TUMOUR TYPES

Cancer genes in selected tumour types.

Lawrence et al. Nature 1‐7 (2014) doi:10.1038/nature12912

MORE COMPLEX ANALYSIS

LIQUID BIOPSY

● The plasma/serum contain several components

● CTCs, exosomes, cfDNA, ctDNA

● To have high levels of cfDNA could be due to several reasons::

Inflammation Wounds Malignant lesions Sport

● Healthy individuals: 3,000 – 5,000 Genomic Equivalents/ml

● Oncologic patients: av 10,000 Genomic Equivalents/ml

Diaz LA & Bardelli A, JCO 2014:32:579‐586

BEAMING DPCR

Pre‐amplification Emulsion PCR Hybridization

Flow Cytometry

Flow cytometry analysis

Mutant signal

MutantDNA

Mutant &Wild-type DNA

Wild-type DNA

Wild

-type

sig

nal

1%

100%

10%

0.1%

0.01%BEAMing

Real-Time PCR

Pyrosequencing

Sanger Sequencing

Detection Capability(mutant DNA/ total DNA)

MIN RESIDUAL DISEASE/EARLY MOLECULAR RECURRENCE

Diehl et at. Nat Med. 2008

After SurgeryDay 244

Mutant signal

Normal signa

l 

Before SurgeryDay of Surgery 

After SurgeryDay 3

Residual Mutant cfDNA

MUTATIONAL LOAD MINIMAL RESIDUAL DISEASE EARLY MOLECULAR RELAPSES

SYSMEX – BASED ON FLOW CITOMETRY

CENTRAL LAB FOR LIQUID BIOPSIES VALENCIA

NEG

NATIONAL NETWORK OF CENTRES USING SYSMEX PLATFORM IN MAIN HOSPITALS. 

OncoBEAM" RAS  CRC Kit

We have data of already 30 patients. Wedetected 69% mutations (KRAS, NRAS)

3mutand beads/0.08% 211 mutand beads/0.46%

NRAS c13

52029 mutand beads/15.8%POS

KRAS c13

10829 mutand beads/12.3%

KRAS c13

NRAS c12

POS

POS

COMPARATIVE

Base calling/Variant calling

Mutation annotationQuerying knowledge base

MassArrayNGS Digital PCR

Clinical interpretation

Clinical report generation

Medical record generation

‐Fit‐for purpose validated pipeline

‐defined performance for variant calling and frequency detection

‐stringent criteria quality control

‐locked down pipeline

‐ Automatic sofware generate variantcalling and frequency detection

‐ Automatic score for quality control‐ Automatic generation of results

AutomaticgenerationOf clinicalreport

Free to decide What has to be included(ethical considerations)

Genetic counseling considerationsReporting variants of unknown significanceReport inclusión criteriaPossible utilization in individual’ls healthcare

Report data inclusion criteria

Manual inspection data

Lockedsystem

Unambigous, clinically relevant interpretation for end user

Fit‐for‐purposeclinical report

Decide data to be shown

COST – EFFECTIVENESS OF A TECHNIQUE IN A SREENING PROGRAM

The higher the multiplexing is, higher the odds that a therapy may be matched

The number of therapies (for now) is finite, so are the number of relevant targets to be asessed

KRAS

PIK3CA

BRAF

FGFR3

EGFR

MEK inhibitor

PI3K inhibitor

BRAF inhibitor

FGFR3 inhibitor

EGFR inhibitor

GENOMIC LANDSCAPE BEFORE AND AFTER ANTI-EGFR THERAPY IN ADVANCED CRC

The population of patients with no MAPK-pathway genomic alterations before treatment (‘all wild-type’) is more likely to respond to epidermal growth factor receptor (EGFR) monoclonal antibodies (mAbs).

The high overlap in primary (left) and acquired (right) resistance mechanisms reinforces that clonal selection is a major determinant of the clinical outcome. Only EGFR mutations have not been identified in pre-treatment lesions. In a substantial proportion of the samples, resistance is polyclonal, with co-occurring RAS mutations and EGFR or PI3K catalytic subunit-α (PIK3CA) mutations. ampl., amplification; mut., mutation.

Dienstmann et al., Nature Reviews Cancer 17, 79–92 (2017)

BREAST CANCER /PRIMARY AND METASTASIS : PIK3CA GENE MUTATIONS

ANTI-EGFR IN ADVANCED CRC

Cejalvo et al,, Breast Can rev Treat, 2015

EMERGING POSITIVE PREDICTIVE BIOMARKERS FOR TREATMENT SELECTION IN ADVANCED CRC

Dienstmann et al., Nature Reviews Cancer 17, 79–92 (2017)

EVOLUTION OF PRECISION MEDICINE PARADIGMS IN COLORECTAL CANCER.

The shift from a clonal perspective for targeted therapies (‘one-gene, one-drug’ and ‘multi-gene, multi-drug’) to a clonal–stromal–immune perspective (‘multi-molecular, multi-drug’) reflects increased understanding of the biology of the disease and advances in biomarker– drug co-development. EGFR, epidermalgrowth factor receptor; IGF1R, insulin-like growth factor 1 receptor; inh., inhibitor; mAb, monoclonal antibody; MSI, microsatellite instability; PD1, programmedcell death protein 1; PIK3CA, PI3K catalytic subunit-α; POLE, DNA polymerase-ε; TGFR, transforming growth factor-β receptor; TKI, tyrosine kinase inhibitor.

PROSPECTS FOR CLINICAL TRANSLATION OF MOLECULAR TESTS IN CRC

Dienstmann et al., Nature Reviews Cancer 17, 79–92 (2017)

Open clinical trials 2016 Active clinical trials

CLINICAL TRIAL ONCOLOGY DEPARTMENT ‐ INCLIVA

0

2

4

6

8

10

12

14

16

18

Fase I Fase II Fase III Fase IIIb

We are participating in a H2020‐Motricolor project withthree clinical trials based on molecular profiling. Patientswill have dedicated therapies according to their genomicprofile.

MEMBERSHIPS FUNDING

Vielen Dank

Cejalvo et al., Breast Cancer Res Treat, 2016